GWAS-identified hyperuricemia-associated IGF1R variant rs6598541 has a limited role in urate mediated inflammation in human mononuclear cells

Gout is a common autoinflammatory joint diseases characterized by deposition of monosodium urate (MSU) crystals which trigger an innate immune response mediated by inflammatory cytokines. IGF1R is one of the loci associated with both urate levels and gout susceptibility in GWAS to date, and IGF-1-IGF-1R signaling is implicated in urate control. We investigate the role of IGF-1/IGF1R signaling in the context of gouty inflammation. Also, we test the gout and urate-associated IGF1R rs6598541 polymorphism for association with the inflammatory capacity of mononuclear cells. For this, freshly isolated human peripheral blood mononuclear cells (PBMCs) were exposed to recombinant IGF-1 or anti-IGF1R neutralizing antibody in the presence or absence of solubilized urate, stimulated with LPS/MSU crystals. Also, the association of rs6598541 with IGF1R and protein expression and with ex vivo cytokine production levels after stimulation with gout specific stimuli was tested. Urate exposure was not associated with IGF1R expression in vitro or in vivo. Modulation of IGF1R did not alter urate-induced inflammation. Developing urate-induced trained immunity in vitro was not influenced in cells challenged with IGF-1 recombinant protein. Moreover, the IGF1R rs6598541 SNP was not associated with cytokine production. Our results indicate that urate-induced inflammatory priming is not regulated by IGF-1/IGF1R signaling in vitro. IGF1R rs6598541 status was not asociated with IGF1R expression or cytokine production in primary human PBMCs. This study suggests that the role of IGF1R in gout is tissue-specific and may be more relevant in the control of urate levels rather than in inflammatory signaling in gout.


GWAS
Gout is a common autoinflammatory joint diseases characterized by deposition of monosodium urate (MSU) crystals which trigger an innate immune response mediated by inflammatory cytokines.IGF1R is one of the loci associated with both urate levels and gout susceptibility in GWAS to date, and IGF-1-IGF-1R signaling is implicated in urate control.We investigate the role of IGF-1/IGF1R signaling in the context of gouty inflammation.Also, we test the gout and urate-associated IGF1R rs6598541 polymorphism for association with the inflammatory capacity of mononuclear cells.For this, freshly isolated human peripheral blood mononuclear cells (PBMCs) were exposed to recombinant IGF-1 or anti-IGF1R neutralizing antibody in the presence or absence of solubilized urate, stimulated with LPS/ MSU crystals.Also, the association of rs6598541 with IGF1R and protein expression and with ex vivo cytokine production levels after stimulation with gout specific stimuli was tested.Urate exposure was not associated with IGF1R expression in vitro or in vivo.Modulation of IGF1R did not alter urate-induced inflammation.Developing urate-induced trained immunity in vitro was not influenced in cells challenged with IGF-1 recombinant protein.Moreover, the IGF1R rs6598541 SNP was not associated with cytokine production.Our results indicate that urate-induced inflammatory priming is not regulated by IGF-1/IGF1R signaling in vitro.IGF1R rs6598541 status was not asociated with IGF1R expression or cytokine production in primary human PBMCs.This study suggests that the role of IGF1R in gout is tissue-specific and may be more relevant in the control of urate levels rather than in inflammatory signaling in gout.
Gout is an important inflammatory disease with high prevalence in developed countries among men and postmenopausal women 1,2 with prevalence increasing worldwide 3 .The precondition for developing gout is the deposition of monosodium urate (MSU) crystals in the joint and other tissues as a result of elevated serum urate levels (hyperuricemia) 4 .While most research focuses on inflammation due to MSU crystal deposition [5][6][7] , there is evidence that soluble urate also increases pro-inflammatory cytokine production 8 , associating hyperuricemia with a hyper-inflammatory state 9,10 , highlighting its pro-inflammatory effects within the intracellular setting together with an altered epigenetic landscape 11 .Additionally, there exist indication on how soluble urate induce epigenetic modifications in myeloid cells, leading to an enhanced inflammatory response 12,13 .Moreover, hyperuricemia may

Participants
The participants in this study consisted of patients with gout (n = 116), hyperuricemic controls (n = 78) and normouricemic volunteers (n = 174), followed at the Rheumatology Department of the "Iuliu Haţieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania.Subjects were enrolled after written informed consent.Peripheral blood was drawn from the cubital vein on EDTA tubes under sterile conditions.The patient study was approved by the Ethical Committee of the "Iuliu Hațieganu" University of Medicine and Pharmacy, Cluj-Napoca (approval no.425/2016) and all participants provided written informed consent.Experiments were conducted according to the principles expressed in the Declaration of Helsinki.All study participants in the gout group were included if they corresponded to the ACR/EULAR 2015 classification criteria with a score of ≥ 8.The cutoff for the asyptomatic hyperuricemia group was a serum urate concentration of 7 mg/dl and negative history of gout flares.The described groups were similar in age and BMI.The gender distribution shows a higher number of men in the gout study group, consistent with the higher prevalence of gout in males.

PBMC isolation and stimulation
Isolation and stimulation of human peripheral blood mononuclear cells (PBMCs) was assessed as described previously 8 .Briefly, PBMCs were separated using Ficoll-Paque and resuspended in RPMI culture medium with Dutch modification (Gibco), supplemented with human pooled serum.Monocyte isolation was done using hyperosmotic Percoll ® solution 28 .Cells were incubated for 24 h with culture medium as negative control, and urate in different concentrations.After priming, culture medium was removed, the remaining adherent cells were washed with warm PBS, then restimulated with medium or LPS with or without MSU crystals.IGF1R was induced by the addition of IGF-1 (R&D Systems, Abingdon, United Kingdom) in concentration of 5 μg/ml.In separate experiments IGF1R signaling was blocked with anti-IGF1R antibody (R&D Systems).

Qantitative PCR for mRNA expression of IGF1R
PBMCs were incubated with the stimuli as mentioned above and after 24 h of incubation at 37 °C in 5% CO 2 .The plates were centrifuged, the supernatant was collected and the cell pellets were lysed with 300 μl of TRIzol Reagent (Invitrogen).RNA purifcation was performed according to manufacturer's instructions.Subsequently, RNA concentrations were determined using NanoDrop software.Isolated RNA was subsequently transcribed into complementary DNA using High-Capacity cDNA Reverse Transcription Kit (Applied BioSystems) followed by quantitative PCR using the Sybr Green Method.The following primers were used in the reaction: IGF1R forward 5′-TCG ACA TCC GCA ACG ACT ATC-3′ and reverse 5′-TCG ACA TCC GCA ACG ACT ATC-3′ and β2-microglobulin forward 5′-ATG AGT ATG CCT GCC GTG TG-3′ and reverse 5′-CCA AAT GCG GCA TCT TCA AAC-3′.Results are shown as fold change in mRNA levels in stimulated samples compared to controls.

Genotyping for IGF1R rs6598541
Three independent groups were genotyped (gout group N = 116; hyperuricemic group N = 78 and healthy volunteers group N = 174).Genomic DNA was isolated from whole blood (Promega) and genotyping was performed on an Illumina Infinium HD assay platform using The Infinium Global Screening Array-24 v3.0 BeadChip.The quality control protocol for genotyping data was performed Using Illumina's GenomeStudio.The SNPs with < 95% call rate were excluded and all the SNPs with 95-98% call rate were verified and manually re-clustered or removed when necessary.The data was exported to PLINK format and further filters were applied: minor allele frequency > 0.01; hardy-weinberg equilibrium test p value > 10 -6 ; samples with heterozygosity rate of ± 3 standard deviations and related individuals were excluded.For the final step, the strands were flipped and all the data was verified to align to the GRCh37 hg19 build.The genotypes generated were checked using a predesigned TaqMan SNP genotyping Assay (Applied Biosystems).

Transcriptomics
Freshly isolated PBMCs were frozen in TRIzol Reagent (Invitrogen) and stored at − 80 °C and were later used for commercial RNA-Seq analysis (Beijing Genomics Institute, BGI, Beijing, China).The integrity of extracted RNA was assessed using Agilent 2100 Bioanalyzer.Oligo dT magnetic beads were used to capture mRNA from total RNA.Purifed ligation products were enriched using PCR amplifcation followed by denaturation and cyclization of ssDNA by splint oligos and DNA ligase generating DNA nanoballs (DNBs).Sequencing of DNBs was performed on DNBseq platform.

Flow cytometry analysis
50 µl fresh EDTA whole blood and antibody solution were mixed by vortex for 4 s, and followed by 10 min incubation in room temperature avoiding light.Afterwards 1 ml NH 4 Cl lysing solution (BD Pharm lyse, BD Biosciences) were added to the stained sample, and vortexed for 5 s.Then the solution was incubated at room temperature for 10 min without light.300 µl of the lysed sample was used for flow cytometry measurement (Beckman Coulter).The antibody solution contained 5 antibodies, including IGF-1R (APC, Life Technologies), CD14 (FITC, Agilent technologies), CD16 (PE, eBioscience), CD45 (PECy7, Beckman Coulter), HLA-DR (PB, Beckman Coulter).Monocytes were first gated by forward scatter and side scatter, and then by CD45.The geometric mean of IGF-1R was used for t-test analysis.

The effect of urate on the expression of IGF1R
To assess the effect of urate on the expression of IGF1R, mRNA was measured after stimulation of PBMCs of eight healthy donors with different concentrations of urate for 24 h.No differences were observed in the expression of the receptor itself (Fig. 1A).In addition, we assessed the mRNA level of the receptor in PBMCs from gout patients in the experimental setup of 24 h urate priming followed by stimulation with LPS 10 ng/ml (TLR4 ligand) for another 24 h.In line with the previous results, no differences regarding expression of IGF1R were observed after stimulation of the cells (Fig. 1B).In contrast, we could observe an increased steady-state mRNA expression of IGF1R in unstimulated monocytes originating from gout patients compared to normouricemic controls (Fig. 1C).

Activation or inhibition of IGF1R does not modify cytokine production in urate priming or trained immunity experiments
Next, we studied the possible contribution of IGF1R to inflammation in an experimental setup consisting of 24 h exposure to recombinant IGF-1 protein (Fig. 2A) (R&D Systems) or anti-IGF1R neutralizing antibody (Fig. 2B) in the presence or absence of solubilized urate, followed by 24 h stimulation with LPS and MSU crystals.Cells treated with solubilised urate (50 mg/dl) together with the recombinant IGF-1 protein (5 ng) produced more IL-1β, but not IL-6 or IL-1Ra compared to control.The cytokine production was not modified by anti-IGF1R neutralizing antibody.Further, we tested if IGF-1 could influence cytokine production in a trained immunity experimental design 29,30 , consisting of stimulation for 24 h with IGF-1 or β-glucan (BG) as positive control, followed by washout and rest for 5 days and subsequent second stimulation with LPS (10 ng/ml).We did not observe an enhanced training effect by IGF1 (Fig. 2C).Bekkering et al. reported SNPs in IGF1R (rs150571637, rs9672558, rs34428109, rs1573891) associated with trained immunity in response to Bacille Camette-Guerin (BCG) and beta-glucan isolated from C. albicans 31 .Thereafter we assessed IGF1R rs1573891 in our study and found no association of this SNP to priming of PBMCs by soluble urate (Fig. 2D).

IGF1R rs6598541 SNP and IGF1R expression levels in freshly isolated PBMCs
We further examined the IGF1R SNP rs6598541, previously associated with serum urate levels 21,23 and gout 24 , for association with IGF1R expression in patients with gout, hyperuricemia and normouricemic controls.The basal expression level of IGF1R in freshly isolated PBMCs from the three groups (Fig. 3A-C) was not associated with rs6598541.Additionally, flow cytometric assessment of IGF1R surface expression on unstimulated PBMCs from healthy donors was also not association with IGF1R rs6598541 (Fig. 3D).

IGF1R rs6598541 SNP and cytokine production in stimulated PBMCS
To further study the possible implication of rs6598541 on inflammation, we assessed the ex vivo cytokine secretion by freshly isolated PBMCs challenged with various stimuli in association with the IGF1R rs6598541 genotypes: cytokine production in response to 24 h stimulation with MSU, palmitate (C16.0) or the combination of MSU crystal with C16.0 (Fig. 4A); urate priming for 24 h and stimulation with LPS for 24 h (Fig. 4B); or 24 h stimulation with LPS 100 ng (Fig. 4C).No significant association was found between the SNP and ex vivo cytokine production (IL-6, IL-1β or IL-1Ra).The same analysis of data obtained following the same experiment carried out for the hyperuricemia individuals and for patients with gout(both active and non-active form) also showed no significant differences for these conditions between genotypes (supplementary Figs. 1, 2, 3).

Discussion
IGF-1 is known to have modulatory roles in human immune responses and a regulatory role in the activation of the peripheral monocytes 26,32 .Interestingly, IGF-1 has dual roles, being involved in inflammation in a context-dependent mode, presenting both pro-and anti-inflammatory properties.After myocardial infarction, IGF-1 acted like an anti-inflammatory cytokine on myeloid cells in vitro while negating the pro-inflammatory phenotype of neutrophils and macrophages in vivo 33 .Also, several in vitro studies revealed anti-inflammatory properties of IGF-1 on astrocytes and microglia 34,35 , whereas another study focused on it's role in decreasing the release of IL-1Ra and increasing IL-1β 36 .
The IGF-1 pathway has recently been shown to be involved in the induction of trained immunity.Since innate immune memory is increasingly shown to be important for urate-induced inflammation, in the present study, we were interested in assessing IGF-1/IGF1R signaling in urate priming using primary PBMCs.
Our data indicate that IGF-1 does not contribute to urate-induced inflammation and blocking IGF1R did not influence the inflammatory responses triggered by urate.We show that urate does not modulate the expression of IGF1R itself in vitro in PBMCs from healthy donors nor in vivo in PBMCs from gout patients.
However, we noticed an increased steady-state mRNA expression of IGF1R in unstimulated PBMCs from gout patients.Increased expression of IGF1R was also seen in leukocytes of rheumatoid arthritis patients, which was associated with systemic inflammation and pain 37 .Interestingly, RA patients with high IGF1R gene expression values were found to have low IGF-1 serum concentrations 37 .In contrast, elevated circulating IGF-1 concentrations were reported in patients with gout and insulin resistance 38 .
Bekkering et al. describe four SNPs in IGF1R associated with differential cytokine production in monocytes trained with β-glucan or BCG vaccine (Bacillus Calmette-Guérin) 25 .We have also assessed one of these four SNPs with respect to urate priming and found no association of these variants to changes in cytokine profiles after urate exposure (Fig. 2D).Therefore, our data provide no evidence for a role of IGF1R in urate-mediated inflammatory priming.
We explored association of the IGF1R rs6598541 SNP, identified as a genetic susceptibility variant for gout and serum urate levels 21,23,24 , with cytokine production in human PBMCs.IGF1R genetic variants are potentially functionally relevant in gout and hyperuricemia since the genetic control of urate levels and risk of gout at the  IGF1R locus also colocalizes with genetic control of IGF1R expression data 23 .For IGF1R rs6598541, the gout risk and elevated serum urate associated allele A is associated with lower IGF1R expression in heart tissue (left ventricle) 39 .In our study, when assessing freshly isolated PBMCs from patients with gout or controls we did not observe association of IGF1R gene expression with rs6598541.In addition, IGF1R surface expression evaluated by flow cytometry in monocytes of healthy donors also did not show modified IGF1R expression associated with rs6598541 genotype.In line with this, ex vivo cytokine secretion by freshly isolated PBMCs challenged with certain stimuli was also not associated with rs6598541.This indicates that this SNP may exert relevant functional roles in gouty inflammation, but they may be tissue specific and mononuclear cells do not show IGF1R expression patterns nor inflammatory cytokine production capacity in relationship to this SNP.
However, data on the role of IGF-1 on urate control give more mechanistic insight.A genetic variant associated with elevated IGF-1 levels (rs35767) correlated to diminished serum urate levels and higher uricosuria 40 .Exposure of HEK293 human embryonic kidney cells to IGF1 resulted in a dose-dependent increase of secretory urate transporters MRP4, NPT1 and ABCG2 and a simultaneous reduction of GLUT9 expression at both the mRNA and protein levels 40 .GLUT9a is the chief transporter for basolateral exit of reabsorbed urate into blood 41 .As reported by Kottgen et al., the urate-associated IGF1R rs6598541 variant is also associated to lower fractional excretion of urate, supporting a role of the IGF1-IGF1R axis on urate transport.This is supported by data showing that, in Xenopus laevis oocytes expressing human IGF1R and urate transporters, IGF-1 promotes urate uptake via IGF1R 39 .Therefore IGF1R association to gout is most probably exerted via urate control rather than inflammatory mechanisms, via IGF1R, GLUT9 expression and activation, leading to urate reabsorbtion 42 .
Another mechanism linking IGF1R rs6598541 to urate levels is insulin resistance.IGF1R rs6598541 was reported to be genome-wide associated with fasting glucose: people without diabetes carrying the A-allele have increased fasting glucose levels, suggesting an association with insulin resistance 43,44 .It is known that hyperuricemia might be contributed to the effect of insulin on decreasing renal urate clearance and sodium excretion in individuals 42,45,46 .In conclusion, our data do not support a role of IGF-1/IGF1R signaling in soluble urate-induced inflammation in primary PBMCs.Consistent with this, functional data associated to genetic variants in IGF1R shows no association with ex vivo cytokine production.

Figure 1 .
Figure 1.Urate effects on mRNA expression in vitro and basal expression of IGF1R of the studied groups.(A) mRNA expression of freshly isolated PBMCs originating from healthy donors (n = 8) treated with different concentrations of uric acid (UA).(B) mRNA expression of freshly isolated PBMCs originating from gout patients (n = 50) treated with UA for 24 h and restimulated with LPS 10 ng/ml.Repeated measurements one-way ANOVA and Tukey's multiple comparisons test, *p < 0.05.(C) mRNA expression of freshly isolated PBMCs originating from healthy controls (n = 113), hyperuricemic patients (n = 19) and gout patients (n = 72).The data is represented as normalized counts.Brown-Forsythe and Welch ANOVA, *p < 0.05.

Figure 4 .
Figure 4. Correlation of the rs6598541 SNP with ex vivo cytokine production.(A) Freshly isolated PBMCs originating from healthy controls (n = 187) stimulated with RPMI, MSU 300 mg/dl, C16, C16/MSU 300 mg/dl for 24 h.After 24 h the supernatants were collected and IL-1β, IL-6 and IL-1Ra (R&D Systems, Minneapolis) was measured.(B) Concentration IL-1β, IL-6 and IL-1Ra measured in the supernatants of PBMCs after stimulation with uric acid of conc.50 mg/dl and 12.5 mg/dl for 24 h, followed by restimulation with LPS 10 ng/ ml.(C) IL-1β, IL-6 and IL-1Ra levels measured in the supernatants of PBMCs after stimulation with LPS 100 ng for 24 h.Graphs depict means ± SEM.